Efeito de microvesículas de melanoma na polarização de neutrófilos humanos

Abstract

The understanding of the mechanisms involved in tumor growth runs through the acknowledgement on its microenvironment, formed by extracellular matrix components, growth factors, cytokines and different cell types. Immune cells present in tumor microenvironment have their pro-inflammatory functions modified to support tumor growth. Recent studies have provided evidence on two phenotypes of tumor-associated neutrophils (TAN): a protumoral, TAN N2, or an antitumoral, TAN N1. Evidence also show that tumor microenvironment is rich in microvesicles (MV), produced abundantly by the tumor. Most MV can not only carry molecular information from their cellular origin but can also modulate the activity of target cells. Although tumor-derived MV were shown to interact with different cells in the microenvironment, there are no reports on its contribution to modulate TAN. Now, we investigate whether MV produced by a human melanoma cell line modulate human neutrophil activity, shifting it to a N2-like phenotype. MV were obtained from conditioned media of human melanoma cell line MV3 cultures and quantified by ZetaView® real-time analyzes and annexin-V staining. Human neutrophils (PMN), isolated by Percoll gradient, were incubated in the presence or absence of MV (10% v/v) or LPS (10 µg/mL) at different times. Chemotaxis was assayed in a 48-wells modified Boyden Chamber. Apoptosis was assessed by flow cytometry. Neutrophil extracellular traps (NETs) formation was evaluated by DNA quantification and immunofluorescence. Intracellular ROS, NO and peroxynitrite production were measured through the oxidation of CM-H2DCFDA, DAF-FM and HPF probes, respectively. Viability of MV3 cells co-cultured with PMN was assessed by MTT assays. Molecular markers gene expression were obtained by qRT-PCR. CXCR4 and arginase contents were obtained by cytometry and immunoblotting, respectively. MV induce PMN chemotaxis, and this effect was inhibited by pertussis toxin and is dependent on PI3K-AKT pathway activation. It was also observed that MV effect on PMN chemotaxis relies on CXCR2, once its inhibitor, SB225002, abrogated PMN migration. MV delayed PMN spontaneous apoptosis, triggered NETs formation and induced intracellular ROS production. Treatment with MV decreased NO basal production, and increased CXCR4 and arginase expression, as well as augmented the mRNA content of several TAN N2 molecular markers, such as arginase, CXCR4 and VEGF in PMN. Finally, PMN treated with MV did not affect melanoma cells viability in co-culture. These data indicate that melanoma-derived MV may have an important role in shaping the phenotype of PMN in tumor microenvironments towards a TAN N2 phenotype, which shows an increased expression pro-tumor molecular markers and presents low cytotoxic properties.